The present invention relates to the isolation of a CAI resistance (CAIR) gene that encodes a protein conferring cellular resistance to carboxyamido-traizole (CAI) and functionally equivalent compounds. This invention further relates to the CAIR protein encoded by the CAI resistance gene, antibodies specific to the protein, and nucleic acid probes that specifically hybridize to the gene. Additionally the invention provides assays for determining CAI resistance in cells, and assays to screen for compositions that obviate CAI resistance. The invention also provides for cell fines that express the CAIR gene and are capable of growing and proliferating in cultures chronically exposed to CAI and functionally equivalent compounds.
Calcium homeostasis is important in the regulation of cellular behavior as it is paramount in regulation of signaling events as well as other cellular and molecular functions. (Cole and Kohn, Cancer and Metastasis Rev. (1993). Carboxyamido-triazole (CAI) is an inhibitor of stimulated calcium influx through voltage gated and nonvoltage-gated calcium influx. It has been shown to inhibit important downstream signaling events including the release or arachidonic acid, production of inositol triphosphate in response to phosphorylation and activation of phospholipase A2, and tyrosine phosphorylation in response to receptor activation (Felder, et al., J. Pharm. Exp. Therap., 257:967-971 (1991); Gusovsky, et al., J. Bio. Chem., 268:7768-7772 (1993)). These signaling pathways regulate proliferation and other cellular events including adhesion, migration, and production of proteases. (Kohn, et al., Cancer Res. (1994) in press, Kohn, et al., Proc. Natl. Acad. Sci. USA, communicated; Kohn and Liotta, J. Nat. Cancer Inst., 82:54-60 (1990)).
Carboxyamido-tiazole (CAI) has been observed to inhibit malignant proliferation, invasion, and metastasis of cancer cells, suggesting the role of CAI, and related compounds, as potential cancer therapeutics (see copending patent application U.S. Ser. No. 07/985,402). Of concern in the development and utilization of cancer therapeutics is the development of resistance by tumor cells to the particular pharmacological regimen to which they are exposed.
Where such a resistance appears it is desirable to ascertain the resistance in order to devise therapeutics that obviate or prohibit the appearance of such resistance. When the resistance is associated with altered gene expression, isolation of the gene encoding proteins associated with the appearance of the resistance not only aids in the elucidation of the mechanism of drug resistance, but also provides useful markers for detecting the acquisition of the resistance as well as useful targets for intervention. The present invention provides an isolated CAI resistance (CAIR) gene whose expression is correlated with CAI resistance. This invention also provides for the protein encoded by this gene. The isolation of this gene and protein provide the aforementioned and other advantages.
The present invention provides for an isolated human nucleic acid encoding a human carboxyamido-triazole resistance (CAIR) protein where the nucleic acid is capable of hybridizing specifically to a second nucleic acid consisting of the nucleic acid sequence of SEQ ID NO:1 in the presence of a human genomic library under stringent conditions. Specifically this nucleic acid comprises the nucleotide sequence of SEQ ID NO:1. More particularly, the isolated nucleic acid, when found in a cell line, expresses the protein it encodes at higher levels when the cell line is cultured in the presence of at least 10 xcexcM concentration of CAI than when the cell line is cultured under the same culture conditions without CAI.
The invention also provides for an isolated human nucleic acid sequence encoding a CAIR protein, wherein the nucleic acid sequence has at least 85%, more particularly at least 95% sequence identity with the nucleic acid of SEQ ID NO:1. The isolated human nucleic acid sequence, when found in a cell line, expresses the protein it encodes at higher levels when the cell line is cultured in the presence of at least 10 xcexcM concentration of CAI than when the cell line is cultured under the same culture conditions without CAI.
This invention additionally provides for an isolated human nucleic acid encoding a CAIR protein having at least 80%, more particularly at least 95% amino acid identity with the amino acid sequence of SEQ ID NO:2. This isolated human nucleic acid, when found in a cell line, expresses the protein it encodes at higher levels when the cell line is cultured in the presence of at least 10 xcexcM concentration of CAI than when the line is cultured under the same culture conditions without CAI.
In another embodiment, this invention provides for an isolated CAIR protein that has at least 80% sequence identity, more particularly at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2. This isolated CAIR protein, when expressed in a cell line, is expressed at higher levels when the cell line is cultured in the presence of at least 10 xcexcM concentration of CAI than when the cell line is cultured under the same culture conditions without CAI.
This invention provides for an isolated CAIR protein, wherein the protein specifically binds to an antibody generated against an immunogen consisting of the amino acid sequence depicted by SEQ ID NO:2. More specifically the carboxyl terminus of the protein consists of a polypeptide of SEQ ID NO:2. In addition, the protein may be recombinantly produced. This protein is expressed at higher levels in a cell line cultured in the presence of at least 10 xcexcM concentration of CAI than in the cell line cultured under the same culture conditions without CAI.
In still another embodiment, this invention provides for an isolated nucleic acid encoding a CAIR resistance protein, wherein the protein specifically binds to an antibody generated against an immunogen consisting of the amino acid sequence depicted by SEQ ID NO:2. More specifically this nucleic acid comprises the nucleotide sequence of SEQ ID NO:1.
In yet another embodiment, this invention provides for cells capable of growing and proliferating when cultured in the presence of CAI ranging in concentration from 1 xcexcM to 45 xcexcM, more particularly in CAI ranging in concentration from 20 xcexcM to 45 xcexcM, and still more particularly in CAI ranging in concentration from 40 xcexcM to 45 xcexcM. Even more particularly these cells are A2058-20R cells or OVCAR3-R cells.
This invention additionally provides for a method of determining resistance of CAI of a biological sample where the method comprises the steps of a) contacting a binding agent capable of specifically binding a CAI resistance protein to the biological sample; b) incubating the binding agent with the biological sample to form a binding agent:CAIR protein complex; and c) detecting the complex. More particularly, the binding agent is an antibody that is specifically immunoreactive with the CAI resistance protein. Even more particularly, the step of detecting comprises a) contacting the complex with a labeled antibody that specifically binds the binding agent; and b) detecting the labeled antibody.
In another embodiment, this invention provides for a method of determining resistance of CAI of a biological sample where the method comprises the steps of a) contacting a binding agent capable of specifically binding nucleotide sequence that encodes a CAI resistance protein with the biological sample; b) incubating the binding agent with the biological sample to form a binding agent:nucleic acid complex; and c) detecting the complex. More particularly, the binding agent is a nucleic acid that hybridizes specifically to a second nucleic acid sequence that encodes a CAI resistance protein under stringent conditions. Even more particularly, the nucleic acid hybridizes specifically to a DNA sequence of SEQ ID NO:1. Still more particularly, the step of detecting comprises detecting a labeled nucleic acid.
This invention provides for a kit for determining the resistance to CAI where the kit comprises a container containing a binding agent capable of specifically binding a CAIR protein. More particularly, the binding agent of the kit is an antibody that specifically binds to a CAIR protein. Even more particularly, the kit further comprises a container containing a means for detecting the antibody.
In another embodiment, the invention provides for a kit for determining resistance to CAI of a biological sample where the kit comprises a container containing a binding agent capable of specifically binding a nucleic acid sequence encoding a CAI resistance protein. More particularly, the binding agent is a second nucleic acid that hybridizes specifically to the nucleic acid sequence under stringent conditions.
The invention also provides for a kit for assaying compounds for a CAI resistance obviating activity, where the kit comprises a container containing a CAI resistant cell. More particularly the cell is A2058-20R or OVCAR3-R.
The invention provides for an antibody that specifically binds to a CAIR protein. More particularly, these antibody is generated against an immunogen consisting of the amino acid sequence depicted by SEQ ID NO:2.
In another embodiment, this invention provides for a pharmacological composition that reduces CAI resistance said composition comprising a binding agent that binds to a nucleotide encoding a CAIR protein. More particularly the binding agent is an antisense molecule that hybridizes specifically to a nucleotide encoding a CAIR protein. Even more particularly the nucleotide encoding a CAIR protein is SEQ ID NO:1.